Chromatography is a physical separation technique that separates the components of a mixture based on their interactions with two phases - a stationary phase and a mobile phase. Mikhail Tswett discovered chromatography in 1906 while separating plant pigments. Chromatography has many applications and has contributed to 12 Nobel prizes between 1937-1972. There are several types of chromatography classified by the mobile phase (liquid, gas), stationary phase (thin layer, paper, column), or separation force (adsorption, partition, ion exchange, gel filtration, affinity). High performance liquid chromatography uses high pressure to allow for faster separation over minutes versus hours with other techniques. Chromatography techniques like thin layer chromatography, gas chromatography, and HPLC are used in various

1. Chromatography, HPLC
Dr.ANSHUL

2. History
Mikhail Tswett, Russian,
Botanist
In 1906 used chromatography to
separate plant pigments.
He called the new technique
chromatography because the
result of the analysis was
'written in color' along the
length of the adsorbent column.
Chroma means “color” and
graphein means to “write”

3. Importance
Chromatography has application in every
branch of the physical and biological sciences
12 Nobel prizes were awarded between 1937 and
1972 alone for work in which chromatography
played a vital role

4. Chromatography is a physical method of
separation in which the components to be
separated are distributed between two
phases
one of which is stationary (stationary phase)
while the other (mobile phase) moves in a
definite direction.
The chromatographic process occurs due to
differences in the distribution constant of the
individual sample components.

5. Chromatography
Technique used to separate and identify
the components of a mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.
Molecules that spend most of their time in
the mobile phase are carried faster.

6. Classification of chromatography
according to mobile phase:
1- Liquid chromatography: mobile phase is
a liquid. (LLC, LSC).
2- Gas chromatography : mobile phase is a
gas. (GSC, GLC).

7. Classification according to the packing of the
stationary phase:
1- Thin layer chromatography (TLC): stationary
phase is a thin layer supported on glass,
plastic or aluminium plates.
2- Paper chromatography (PC): stationary
phase is a thin film of liquid supported on an
inert support.
3- Column chromatography (CC): stationary
phase is packed in a glass column.

8. Classification according to the force of
separation:
1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.

9. Separations in TLC involve distributing a mixture of
two or more substances between a stationary
phase and a mobile phase.
The stationary phase:
a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate.
The mobile phase:
a developing liquid which travels up the stationary
phase, carrying the samples with it.
Components of the samples will separate on the
stationary phase according to-
how much they adsorb on the stationary phase
versus how much they dissolve in the mobile
phase.
Thin Layer Chromatography (TLC)

10. Thin Layer Chromatography (TLC)

11. Procedure
A TLC plate is a sheet of glass, metal, or plastic which is
coated with a thin layer of a solid adsorbent (usually
silica or alumina).
A small amount of the mixture to be analyzed is spotted
near the bottom of this plate.
The TLC plate placed in a shallow pool of a solvent in a
developing chamber so that only the very bottom of
the plate is in the liquid.
This liquid, mobile phase, slowly rises up the TLC plate
by capillary action.

12. As the solvent moves, an equilibrium is
established for each component of the mixture
between the molecules of that component
which are adsorbed on the solid and the
molecules which are in solution.
components differ in solubility and in the
strength of their adsorption to the adsorbent
so components will be carried farther up on
the plate than others.
When the solvent has reached the top of the
plate, the plate is removed from the
developing chamber, dried, and the separated
components of the mixture are visualized.

13. Identification of the Spots
If the spots can be seen, outline them
with a pencil.
If no spots are obvious after staining-
hold the plate under a UV lamp
Many organic compounds can be
seen using this technique.
many commercially made plates also
contain a substance which aids in
the visualization of compounds.

14. Visualizing Agents
Ninhydrin- Amino acids:
Rhodamine B- Lipids.
Antimony trichloride- Steroids
Aniline phthalate- Sugar.

15. Interpreting the Data
The Rf (retention factor) value for each spot
should be calculated.
It is characteristic for any given compound on
the same stationary phase using the same
mobile phase.
Hence, known Rf values can be compared to
those of unknown substances to aid in their
identifications.

18. Application
• Identification of drugs in urine.
• Analysis of amino acids & carbohydrates in urine.
• Analysis of lipids in amniotic fluid.(L/S ratio in RDS.)
Limitation-
Two different substance may exhibit same Rf value.

19. Paper Chromatography
A method of partition chromatography using filter paper
strips as carrier or inert support.
The factor governing separation of mixtures of solutes on
filter paper is the partition between two immiscible
phases.
One is usually water adsorbed on cellulose fibres in the
paper (stationary phase).
The second is the organic solvent flows and migrate the
sample on the paper (mobile phase).

20. Paper Chromatography

21. Paper Chromatography
Application-
• Detects amino acid, sugar and pigments.
• Identification of type of aminoaciduria-
Cystinuria, PKU.

22. Gas Liquid Chromatography
• Type of partition chromatography used for
qualitative and quantitative analysis of large
no. of compounds.
• It provides high speed of resolution very
good reproducibility and high sensitivity.
• It is based on partition of a compound
between liquid and gas phase.

23. Gas Liquid Chromatography
• Principle :
–Volatile molecule (lowest boiling point)
swept rapidly along with carrier gas.
–Molecules soluble in liquid phase – retained
in liquid .
Volatility and solubility affect – rate of flow of
sample molecule in gas column.

24. Gas - Liquid Chromatography
Sample introduced by
syringe.
Column separates components.
(Heated in oven)
Detector monitors compounds
emerging from outlet.
Recorder plots signals as
a chromatogram.

25. Uses Of GLC
• Volatile organic compound separation.
• Non volatile substance separation (converting
into volatile by oxidation, acylation, alkylation)
• Alcohol, ester, fatty acid and amines present in
biological sample can be separated.
• Concentration of individual element such as
carbon and hydrogen - determined accurately.
• Functional group can be identified.
• Position of double bond can be determined by
cleaving the bond by oxidation.

26. Adsorption Chromatography
Principle :
• It is differential adsorption of solute on the
surface of stationary phase.
• Certain solid material have ability to hold
molecule at their surface.
• This adsorption forces involves weak & non
ionic forces such as hydrogen, Vanderwal’s
forces.
• This is used for separation of organic
compound.

27. Adsorption Chromatography
Adsorbent :
• Should be inert (should not react with eluting solvent).
• Should be stable for long peroid.
• Should be cheap.
Most commonly used adsorbent are :
• Silica gel- AA and steroids.
• Alumina -small organic molecule .
• Activated carbon –carbohydrate and protein
Most strongly adsorb component forms top most band
while least adsorb component forms lower most band on
adsorbent media

28. Adsorption Chromatography
Factors Affecting :
• Nature of adsorbent.
• Nature of solvent.
• Rate of flow of solvent through column.
• Dimension of column.
• Temperature of column during separation.

29. Adsorption Chromatography
Applications :
Separation of
• Aromatic compound phenol, amine etc.
• Aliphatic hydrocarbon from aromatic
hydrocarbon
• Nucleic acid

30. Ion Exchange Chromatography
Principle :
Based on exchange of ions b/w charges stationary
phase and ions of opposite charge in mobile phase.
Soluble mixture to be separated, allowed to pass
through a column of ion exchange resins which are
insoluble polymer containing ionic group.
Ion exchange resin made of-cellulose, dextran,
acrylamide polymer etc.

32. Ion Exchange Chromatography
Ion exchange resin : two type
1: Cation exchange resin- contain negatively
charge ions (-carboxyl, -phosphate ions and
bind covalently to positively charge ion in
mobile phase.
2: Anion exchange resin-contain positively
charge ion & binds to negatively charged
ions in mobile phase

33. Ion Exchange Chromatography
Application:
Separation of
– Amino acid
– Glycated hemoglobin
– Oligonucleotides
– Removal of inorganic ions from aqueous mixture.
Disadvantage :
– Different substance with same ionic charge – can
not be separated

34. Gel Filtration Chromatography
Syn.(size exclusion, gel permeation, molecular
exclusion chromatography)
Principle :
Separation based on –molecular size, shape, weight.
A mixture of molecules of different size placed on
top of gel filtration column large molecule pass
through interstitial space b/w beads without any
resistance
Small molecule enter in pores of beads and
effectively removed from stream of eluting solvent.
Movement of small molecule is retarted- eluting out
slowly

35. Gel Filtration Chromatography
Advantage
• Separation of substance - independent of ph,
temperature, ionic strength and buffer
composition.
• No adsorption- labile substance (enzyme) not
affected
• Elution volume- directly related to molecular
weight.

36. Gel Filtration Chromatography
Application :
• Use to purifying enzymes and other
proteins and fractionating nucleic acid.
• To determine molecular weight of
protein.
• Agarose gel – various type of RNA and
viruses are fractionated and purified.

37. Affinity Chromatography
Principle :
Based on specific and non covalent
binding of substance like protein &
enzyme to specific ligand which is
attached to gel matrix.
Ex. Separation of LDH from RBC-
using NAD+ cofactor as ligand which
is linked to affinity gel.

38. Affinity Chromatography
Application :
• Isolation of albumin, DNA polymerase,
coagulation factors.
• Isolation of glycoproteins.
• Purifying human immunoglobulins.
• Purification of bone collagen, HBsAg,
ribosome.

39. High Pressure Liquid Chromatography (HPLC)
Method in which the solution
to be analyzed is passed, under
high pressure, through a long, thin column
packed with tiny beads such that analyses
are completed in minutes rather than hours
and with improved resolution.

40. (HPLC)
• Columns are packed in strong stainless steel
cylinder - withstand high pressure.
• Non compressible resin - used as supporting
media.
• UV absorption, fluorescence, regenerative
index detectors are used.
• All physiochemical mechanism(adsorption,
partition, affinity, ion exchange) are possible
in HPLC.
• Entire HPLC process can be completed in a few
minutes of effective separation and detection.

41. HPLC

42. HPLC
Uses :
• Spectrophotometer detector- identification of
drugs in urine and serum.
• Flurometer- amino acids & other primary amine
can be detected.
• Electrochemical detector- urinary catecholamine
& compounds(unsaturated FAs & PGs) can
analyzed.
• Urinary VMA(vanillylmandelic acid),
metanephrins, homovanillic acid can be
analyzed.

43. THANK YOU